We have used monoclonal antiestrophilin antibodies to develop an improved immunocytochemical method for localizing estrogen receptors in tissue sections with an indirect immunoperoxidase technique. Rat monoclonal antibodies were raised against human estrogen receptor (estrophilin) protein derived from the cytosol of MCF-7 human breast cancer cells. These monoclonal antibodies have been shown to have extensive cross-reactivity with estrogen receptors from various primate and nonprimate tissues. We have used frozen sections of human proliferative phase endometrium and an indirect immunoperoxidase technique to establish conditions for demonstrating estrogen receptor antigenic determinants in frozen tissue sections. The method involves (a) brief fixation with formaldehyde-containing fixatives either prior to freezing or immediately after cutting cryostat sections, (b) bleaching the tissue of endogenous peroxidase activity, (c) application of primary antibody or control immunoglobulin, (d) application of an adsorbed bridging antibody (goat antirat IgG), and (e) application of rat peroxidase-antiperoxidase followed by diaminobenzidine. Specific nuclear staining for estrogen receptor antigenic determinants was observed in the vast majority of epithelial and stromal cells. No specific cytoplasmic staining was identified in cryostat sections of any of the 17 cases studied for this report.